Resistance Determinants and Mobile Genetic Elements of an NDM-1-Encoding <i>Klebsiella pneumoniae</i> Strain

<div><p>Multidrug-resistant <i>Enterobacteriaceae</i> are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-lactamase NDM-1 is rapidly spreading. We present the complete genome of <i>Klebsiella pneumoniae</i> ATCC BAA-2146, the first U.S. isolate found to encode NDM-1, and describe its repertoire of antibiotic-resistance genes and mutations, including genes for eight β-lactamases and 15 additional antibiotic-resistance enzymes. To elucidate the evolution of this rich repertoire, the mobile elements of the genome were characterized, including four plasmids with varying degrees of conservation and mosaicism and eleven chromosomal genomic islands. One island was identified by a novel phylogenomic approach, that further indicated the <i>cps-lps</i> polysaccharide synthesis locus, where operon translocation and fusion was noted. Unique plasmid segments and mosaic junctions were identified. Plasmid-borne <i>bla</i>_CTX-M-15 was transposed recently to the chromosome by IS<i>Ecp1</i>. None of the eleven full copies of IS<i>26</i>, the most frequent IS element in the genome, had the expected 8-bp direct repeat of the integration target sequence, suggesting that each copy underwent homologous recombination subsequent to its last transposition event. Comparative analysis likewise indicates IS<i>26</i> as a frequent recombinational junction between plasmid ancestors, and also indicates a resolvase site. In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected. In a second novel use, circular transposition intermediates were detected for the novel insertion sequence IS<i>Kpn21</i> of the IS<i>NCY</i> family, suggesting that it uses the two-step transposition mechanism of IS<i>3</i>. Robust genome-based phylogeny showed that a unified <i>Klebsiella</i> cluster contains <i>Enterobacter aerogenes</i> and <i>Raoultella</i>, suggesting the latter genus should be abandoned.</p></div

Genomic islands.

a<p>Islands integrated into RNA genes are assigned the orientation of the target gene; remaining islands are arbitrarily assigned the genomic orientation.</p>b<p>Putative <i>att</i> sites identified at each flank.</p>c<p>Status in the closely related <i>K. pneumoniae</i> HS11286 chromosome; No/Yes, absent/present in HS11286; Alt, alternative island in HS11286 at the Kpn2146 island site.</p>d<p>The PHAST islands are strongly phage-like; we note where other islands have proteins suggestive of phage function, and other features of interest.</p>e<p>All islands had phyloblocks support.</p>f<p>Damage denotes loss of an acceptor stem nucleotide in the tRNA gene fragment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Mantri1" target="_blank">[31]</a>; phage names are the closest relatives determined by PHAST.</p>g<p>CP003200.1/581777-594029; with IS<i>903</i> inserted at position 4245 with 9-bp DR; directly at tRNA-Leu <i>att</i> site with no intervening Kpn21L.</p>h<p>50403-bp alternative island at CP003200.1/1288317-1338720 with same initial 1989 bp as Kpn49R.</p>i<p>CP003200.1/1778319-1808718; 2 mismatches; with 7201 bp deleted from peg.320 to 3′ end of gpII intron.</p>j<p>CP003200.1/2277469-2325549; with IS<i>Ec22</i> at position 15471 with 8-bp DR.</p>k<p>CP003200.1/4502805-4558770; with IS<i>903B</i> insert at position 9777 with 9-bp DR.</p>l<p>CP003200.1/4819193-4835187; no mismatches.</p

Learned phyloblocks identify a new island and the highly variable capsular polysaccharide and lipopolysaccharide synthesis gene cluster (<i>cps-lps</i>).

<p>Nonubiquity phyloblocks: those missing in at least one of the 11 reference chromosomes. Complex phyloblocks: those requiring more than one gain/loss event to reconcile the phylotype with the genome tree of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone-0099209-g001" target="_blank">Fig. 1</a>. As a percentage of their combined 411 kbp, the learned phyloblocks mapped either to the training islands (81.9%), the two newly indicated regions (12.0%), insertion sequences (2.1%), or to small scattered regions that did not show hallmarks of islands (4.0%). Red segments: the 11 final islands (including a tandem array of Kpn21L and Kpn11L). Circles, the two newly indicated regions.</p

pKpn2146b.

<p>Key, color coding of genes, mobile elements, and unique regions and juxtapositions, with additional colors for non-gene features. Inner ring, representative long matches to other plasmids. Innermost black arrows, recent recombination events. HR, homologous recombination; abR, antibiotic resistance.</p

Enzymes, efflux pumps, and mutations expected to confer resistance to antibiotics of clinical relevance^a.

a<p>Excluding the resistance enzyme for bleomycin, an antibiotic used clinically only as an antitumor agent.</p>b<p>Variant number from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone-0099209-t001" target="_blank">Table 1</a> of Ramirez <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Ramirez2" target="_blank">[37]</a> is used to distinguish the AAC(6′)-Ib variants.</p>c<p>Two silent differences between the two copies.</p>d<p>Probable efflux substrates identified from literature sources including ARDB; the substrates list is not comprehensive and in many cases has been deduced from organisms other than <i>K. pneumoniae</i>.</p>e<p>Mdt genes are scattered over the chromosome.</p

pNDM-US.

<p>Key, color coding of genes, mobile elements, and unique regions and juxtapositions, with additional colors for non-gene features. Inner ring, representative long matches to other plasmids. abR, antibiotic resistance.</p

<i>Klebsiella</i> phylogeny.

<p>Tree for 108 genomes based on a 2.93-Mbp alignment, rooted at the midpoint of the outgroup (Ecl/Yre) branch. Nodes with <30% bootstrap support were combined forming the multifurcated dashed line; otherwise support values are shown only when <100%. Brackets: Kpn multilocus sequence type (ST). Inset: enlargement of the “core Kpn” phylogeny. Kpn2146 falls in a clade containing fellow ST11 strains Kpn JM45 and Kpn HS11286 and a tight clade (circled) of ST258 and ST512 strains. The ST258/ST512 clade is heavily sequenced, and represented here with only five of its most diverse members. Bold: complete genomes used for phyloblocks analysis. Species name abbreviations: Kpn, <i>K. pneumoniae</i>; Ksp, <i>K. sp</i>.; Kpl, <i>K. cf. planticola</i>; Kox, <i>K. oxytoca</i>; Kva, <i>K. variicola</i>; Eae, <i>Enterobacter aerogenes</i>; Ecl, <i>E. cloacae</i>; Ror, <i>Raoultella ornithinolytica</i>; Yre, <i>Yokanella regensburgei</i>.</p

Replicon copy numbers.

a<p>Unique 21-mers were identified for each replicon, and counted among MiSeq reads.</p>b<p>Mean 21-mer coverages were very similar to median values, and were normalized to that of the chromosome (which had 68.8 occurrences per 21-mer).</p

pKpn2146c.

<p>Key, color coding of genes, mobile elements, and unique regions and juxtapositions, with additional colors for non-gene features. Inner ring, representative long matches to other plasmids. Innermost black arrows, recent recombination events. HR, homologous recombination; abR, antibiotic resistance.</p

Operon translocation and fusion at the <i>cps</i>-<i>lps</i> polysaccharide synthesis locus.

<p>The <i>cps</i> P1, P2 and P3 promoters are taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Arakawa1" target="_blank">[68]</a>, while a promoter (P<i>lps</i>) has been mapped in <i>K. pneumoniae</i> MGH 78578 to the intergenic space between <i>uge</i> and the first <i>lps</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Seo1" target="_blank">[69]</a>. <b>A</b>) The <i>cps-lps</i> region of <i>K. pneumoniae</i> 342, which is typical of <i>Klebsiella</i>. Genes of <i>cps</i> are in yellow (common in most strains) or blue (varying in gene identity, count, and order); genes of <i>lps</i> are in red. The <i>manCB</i> unit (orange arrows) is occasionally found in <i>cps</i>, and occasionally in <i>lps</i>, and here unusually in both. The diamond represents the JUMPstart DNA/RNA motif at whose <i>ops</i> sequence RfaH is loaded onto the elongating RNA polymerase in place of NusG, preventing Rho-based termination for the small number of long transcription units that are controlled by <i>ops</i>-RfaH, and physically coupling the elongating RNA polymerase to the trailing ribosome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Artsimovitch1" target="_blank">[70]</a>. <b>B</b>) Kpn2146 <i>cps-lps</i>. The boxed <i>cps</i> P3 unit has been deleted from its usual site, and moreover translocated to a nearby position, apparently by transposition and/or homologous recombination mechanisms; note the complex pattern of surrounding IS insertions and the directly repeated flanking sequence copies (gray arrows).ΔIS, incomplete IS copy; dotted lines, gene or IS interrupted by ISs; GT, glucosyl transferase, Hyp, hypothetical.</p